Site-Specific Bioconjugation of a Murine Dihydrofolate Reductase Enzyme by Copper(I)-Catalyzed Azide-Alkyne Cycloaddition with Retained Activity

Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) is an efficient reaction linking an azido and an alkynyl group in the presence of copper catalyst. Incorporation of a non-natural amino acid (NAA) containing either an azido or an alkynyl group into a protein allows site-specific bioconjugation in mild conditions via CuAAC. Despite its great potential, bioconjugation of an enzyme has been hampered by several issues including low yield, poor solubility of a ligand, and protein structural/functional perturbation by CuAAC components. In the present study, we incorporated an alkyne-bearing NAA into an enzyme, murine dihydrofolate reductase (mDHFR), in high cell density cultivation of Escherichia coli, and performed CuAAC conjugation with fluorescent azide dyes to evaluate enzyme compatibility of various CuAAC conditions comprising combination of commercially available Cu(I)-chelating ligands and reductants. The condensed culture improves the protein yield 19-fold based on the same amount of non-natural amino acid, and the enzyme incubation under the optimized reaction condition did not lead to any activity loss but allowed a fast and high-yield bioconjugation. Using the established conditions, a biotin-azide spacer was efficiently conjugated to mDHFR with retained activity leading to the site-specific immobilization of the biotin-conjugated mDHFR on a streptavidin-coated plate. These results demonstrate that the combination of reactive non-natural amino acid incorporation and the optimized CuAAC can be used to bioconjugate enzymes with retained enzymatic activityope

Tactual perception: a review of experimental variables and procedures

This paper reviews literature on tactual perception. Throughout this review we will highlight some of the most relevant variables in touch literature: interaction between touch and other senses; type of stimuli, from abstract stimuli such as vibrations, to two- and three-dimensional stimuli, also considering concrete stimuli such as the relation between familiar and unfamiliar stimuli or the haptic perception of faces; type of participants, separating studies with blind participants, studies with children and adults, and an analysis of sex differences in performance; and finally, type of tactile exploration, considering conditions of active and passive touch, the relevance of movement in touch and the relation between exploration and time. This review intends to present an organised overview of the main variables in touch experiments, attending to the main findings described in literature, to guide the design of future works on tactual perception and memory.This work was funded by the Portuguese “Foundation for Science and Technology” through PhD scholarship SFRH/BD/35918/2007

Tactile localization biases are modulated by gaze direction

Identifying the spatial location of touch on the skin surface is a fundamental function of our somatosensory system. Despite the fact that stimulation of even single mechanoreceptive afferent fibres is sufficient to produce clearly localised percepts, tactile localisation can be modulated also by higher-level processes such as body posture. This suggests that tactile events are coded using multiple representations using different coordinate systems. Recent reports provide evidence for systematic biases on tactile localisation task, which are thought to result from a supramodal representation of the skin surface. While the influence of non-informative vision of the body and gaze direction on tactile discrimination tasks has been extensively studied, their effects on tactile localisation tasks remain largely unexplored. To address this question, participants performed a tactile localization task on their left hand under different visual conditions by means of a mirror box; in the mirror condition a single stimulus was delivered on participants’ hand while the reflexion of the right hand was seen through the mirror; in the object condition participants looked at a box through the mirror, and in the right hand condition participants looked directly at their right hand. Participants reported the location of the tactile stimuli using a silhouette of a hand. Results showed a shift in the localization of the touches towards the tip of the fingers (distal bias) and the thumb (radial biases) across conditions. Critically, distal biases were reduced when participants looked towards the mirror compared to when they looked at their right hand suggesting that gaze direction reduces the typical proximo-distal biases in tactile localization. Moreover, vision of the hand modulates the internal configuration of points’ locations, by elongating it, in the radio-ulnar axis

Developmental perspectives on interpersonal affective touch

In the last decade, philosophy, neuroscience and psychology alike have paid increasing attention to the study of interpersonal affective touch, which refers to the emotional and motivational facets of tactile sensation. Some aspects of affective touch have been linked to a neurophysiologically specialised system, namely the C tactile (CT) system. While the role of this sys-tem for affiliation, social bonding and communication of emotions have been widely investigated, only recently researchers have started to focus on the potential role of interpersonal affective touch in acquiring awareness of the body as our own, i.e. as belonging to our psychological ‘self’. We review and discuss recent developmental and adult findings, pointing to the central role of interpersonal affective touch in body awareness and social cognition in health and disorders. We propose that interpersonal affective touch, as an interoceptive modality invested of a social nature, can uniquely contribute to the ongoing debate in philosophy about the primacy of the relational nature of the minimal self

Viewing the body modulates neural mechanisms underlying sustained spatial attention in touch

Cross-modal links between vision and touch have been extensively shown with a variety of paradigms. The present event-related potential (ERP) study aimed to clarify whether neural mechanisms underlying sustained tactile-spatial attention may be modulated by visual input, and the sight of the stimulated body part (i.e. hands) in particular. Participants covertly attended to one of their hands throughout a block to detect infrequent tactile target stimuli at that hand while ignoring tactile targets at the unattended hand, and all tactile non-targets. In different blocks, participants performed this task under three viewing conditions: full vision; hands covered from view; and blindfolded. When the participants' hands were visible attention was found to modulate somatosensory ERPs at early latencies (i.e. in the time range of the somatosensory P100 and the N140 components), as well as at later time intervals, from 200 ms after stimulus onset. By contrast, when participants were blindfolded and, crucially, even when only their hands were not visible, attentional modulations were found to arise only at later intervals (i.e. from 200 ms post-stimulus), while earlier somatosensory components were not affected by spatial attention. The behavioural results tallied with these electrophysiological findings, showing faster response times to tactile targets under the full vision condition compared with conditions when participants' hands were covered, and when participants were blindfolded. The results from this study provide the first evidence of the profound impact of vision on mechanisms underlying sustained tactile-spatial attention, which is enhanced by the sight of the body parts (i.e. hands). © Federation of European Neuroscience Societies and Blackwell Publishing Ltd

Which finger? Early effects of attentional selection within the hand are absent when the hand is viewed

The sight of a hand can bias the distribution of spatial attention, and recently it has been shown that viewing both hands simultaneously can facilitate spatial selection between tactile events at the hands when these are far apart. Here we directly compared the electrophysiological correlates of within-hand and between-hands tactile-spatial selection to investigate whether within-hand selection is similarly facilitated by viewing the fingers. Using somatosensory event-related potentials, we have shown that effects of selection between adjacent fingers of the same hand at early somatosensory components P45 and N80 were absent when the fingers were viewed. Thus, we found a detrimental effect of vision on tactile-spatial within-body part (i.e. hand) selection. In contrast, effects of tactile-spatial selection between hands placed next to each other, which were first found at the P100 component, were unaffected by vision of the hands. Our findings suggest that (i) within-hand and between-hands selection can operate at different stages of processing, and (ii) the effects of vision on within-hand and between-hands attentional selection may reflect fundamentally different mechanisms. © 2010 Federation of European Neuroscience Societies and Blackwell Publishing Ltd

Richtlinien zur Konstruktion von Anti-Vibrations-Systemen fuer unterschiedliche handgefuehrte schlagende Maschinen Endbericht

SIGLEAvailable from TIB Hannover: F95B537+a / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekBundesministerium fuer Forschung und Technologie (BMFT), Bonn (Germany)DEGerman